One feature of pancreatic ductal adenocarcinoma (PDAC) is the fibrotic stroma, which is a result of interactions between tumor cells, immune suppressive cells and inflammatory fibroblasts, arising from `pancreatic stellate cells' (PSC). The stroma is a significant barrier restraining immunity and targeting the tumor microenvironment (TME) can be an innovative means to improve access of effector immune cells to PDAC. This translational proposal addresses the hypothesis that targeting Hsp90 can modulate the composition of the TME, through its effects on cytokine production by PSC, and regulate signal transduction in T cells to improve the efficacy of anti-PD-1 immunotherapy. We show for the first time, that Hsp90 inhibitors (Hsp90i) limit growth and cytokine secretion at the transcriptional level from PSC. In addition, our published data indicate Hsp90i inhibits key pathways and PDAC growth in vitro and in vivo. Finally, we show Hsp90i improves the efficacy of PD-1 blockade in murine PDAC tumor models. These data led to an investigator-initiated Phase Ib/II clinical trial of XL888 (Hsp90i) and pembrolizumab (anti-PD-1) now accruing at our institution. The clinical costs of this trial are committed, thus this proposal will deliver added value through unique correlative studies. In fact, this novel trial will provide paired biopsies and blood to validate hypotheses from preclinical models regarding the impact of Hsp90i on the TME. Pilot data using blood of patients on trial show the functional response of T cells is preserved. RNA seq and MSigDB analysis of sorted, stem-like CD8+ T cells from patients on trial reveals modulation of genes related to STAT and NF-?B signaling that impact T effector cell generation and function. This proposal has three Specific Aims: 1) To evaluate the impact of Hsp90i on PSC and inflammatory fibroblasts in the PDAC TME. Primary PSC obtained from PDAC patients will be used in culture and in organoids to study how pharmacologic or genetic alteration of Hsp90 influences PSC. Paired biopsies from the trial will interrogate the impact of Hsp90i in combination with anti-PD-1 Ab on subsets of PSC or fibroblasts in the TME. 2) To study how Hsp90i can alter immunomodulatory cytokines relevant to PDAC tumors. A panel of established human and murine PDAC cell lines, along with organoids from PDAC patients will be used to evaluate the effect of Hsp90i on chemokine and cytokine production as a means to influence the cellular composition in the TME. To complement these pre-clinical studies, cytokine and chemokine signatures, along with immune suppressor cells will be assessed in patient blood and paired biopsy specimens from the clinical trial before and after treatment. 3) To determine how Hsp90i modulates T cell phenotype and function to enhance efficacy of PD-1 blockade. We will study if Hsp90i limits NF-?B and Jak/STAT signalilng as a mechanism to enhance sensitivity of T cells to PD-1 blockade. The immune activity of Hsp90i will be confirmed using Hsp90i-resistant vs. sensitive tumors, and T cell depleting antibodies in murine models. Effects of anti-PD-1 Ab +/- Hsp90i on key biomarkers will be confirmed in tumor infiltrating lymphocytes (TIL) and immune cells from animal studies and clinical trial samples.